Issue 4, 2024

Sortase mediated protein ubiquitination with defined chain length and topology

Abstract

Ubiquitination is a key post-translational modification on protein lysine sidechains known to impact protein stability, signal transduction cascades, protein–protein interactions, and beyond. Great strides have been made towards developing new methods to generate discrete chains of polyubiquitin and conjugate them onto proteins site-specifically, with methods ranging from chemical synthetic approaches, to enzymatic approaches and many in between. Previous work has demonstrated the utility of engineered variants of the bacterial transpeptidase enzyme sortase (SrtA) for conjugation of ubiquitin site-specifically onto target proteins. In this manuscript, we’ve combined the classical E1/E2-mediated polyubiquitin chain extension approach with sortase-mediated ligation and click chemistry to enable the generation of mono, di, and triubiquitinated proteins sfGFP and PCNA. We demonstrate the utility of this strategy to generate both K48-linked and K63-linked polyubiquitins and attach them both N-terminally and site-specifically to the proteins of interest. Further, we highlight differential activity between two commonly employed sortase variants, SrtA 5M and 7M, and demonstrate that while SrtA 7M can be used to conjugate these ubiquitins to substrates, SrtA 5M can be employed to release the ubiquitin from the substrates as well as to cleave C-terminal tags from the ubiquitin variants used. Overall, we envision that this approach is broadly applicable to readily generate discrete polyubiquitin chains of any linkage type that is accessible via E1/E2 systems and conjugate site-specifically onto proteins of interest, thus granting access to bespoke ubiquitinated proteins that are not currently possible.

Graphical abstract: Sortase mediated protein ubiquitination with defined chain length and topology

Supplementary files

Article information

Article type
Communication
Submitted
27 Nov. 2023
Accepted
01 Febr. 2024
First published
07 Febr. 2024
This article is Open Access
Creative Commons BY-NC license

RSC Chem. Biol., 2024,5, 321-327

Sortase mediated protein ubiquitination with defined chain length and topology

N. R. Raniszewski, J. N. Beyer, M. I. Noel and G. M. Burslem, RSC Chem. Biol., 2024, 5, 321 DOI: 10.1039/D3CB00229B

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